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Structural integration in hypoxia-inducible factors

Abstract

The hypoxia-inducible factors (HIFs) coordinate cellular adaptations to low oxygen stress by regulating transcriptional programs in erythropoiesis, angiogenesis and metabolism. These programs promote the growth and progression of many tumours, making HIFs attractive anticancer targets. Transcriptionally active HIFs consist of HIF-α and ARNT (also called HIF-1β) subunits. Here we describe crystal structures for each of mouse HIF-2α–ARNT and HIF-1α–ARNT heterodimers in states that include bound small molecules and their hypoxia response element. A highly integrated quaternary architecture is shared by HIF-2α–ARNT and HIF-1α–ARNT, wherein ARNT spirals around the outside of each HIF-α subunit. Five distinct pockets are observed that permit small-molecule binding, including PAS domain encapsulated sites and an interfacial cavity formed through subunit heterodimerization. The DNA-reading head rotates, extends and cooperates with a distal PAS domain to bind hypoxia response elements. HIF-α mutations linked to human cancers map to sensitive sites that establish DNA binding and the stability of PAS domains and pockets.

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Figure 1: Overall architectural features of the HIF-2α–ARNT heterodimer and its comparison with CLOCK–BMAL1.
Figure 2: Domain interfaces of the HIF-2α–ARNT complex.
Figure 3: Ligand binding pockets in the HIF-2α–ARNT heterodimer.
Figure 4: DNA-bound HIF-α–ARNT structures.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in Protein Data Bank under accession numbers 4ZP4 (HIF-2α–ARNT apo), 4ZQD (HIF-2α–ARNT–0X3), 4ZPH (HIF-2α–ARNT–Proflavine), 4ZPK (HIF-2α–ARNT–DNA) and 4ZPR (HIF-1α–ARNT–DNA).

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Acknowledgements

We thank Y. Zhang and M. Wang for isolation and identification of trypaflavin.

Author information

Authors and Affiliations

Authors

Contributions

D.W. and F.R. conceived the study; D.W. isolated the proteins, carried out crystallizations and conducted biochemical studies; N.P. produced the expression and mutation constructs; Y.K. and J.L. collected synchrotron diffraction data; D.W., Y.K. and F.R. analysed the data; F.R. and D.W. wrote the manuscript.

Corresponding author

Correspondence to Fraydoon Rastinejad.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 The mammalian bHLH-PAS family of transcription factors.

a, Diagram showing the partner choices for heterodimerization between members of this family. ARNT is ubiquitously expressed, while ARNT2 (paralogue of ARNT) is mainly expressed in the central nervous system6,7. b, Sequence identity comparison among the same domains from mouse ARNT, HIF-1α, HIF-2α, HIF-3α, CLOCK and BMAL1 proteins.

Extended Data Figure 2 Structure of the HIF-2α–ARNT complex in cartoon mode.

Overall structure of HIF-2α–ARNT complex (centre), and individual domains of ARNT (top) and HIF-2α (bottom) with their secondary structures labelled.

Extended Data Figure 3 Comparison of the overall structures of HIF-2α–ARNT and CLOCK–BMAL1 complexes.

Two complexes are superposed by aligning the bHLH domains (a) or PAS-B domains (b) of ARNT and BMAL1, respectively. Colours used in labels match those used in figures for the same components.

Extended Data Figure 4 Domain interfaces of the HIF-2α–ARNT complex and the comparison with other structures.

a, Overall structure of HIF-2α–ARNT complex with domain interfaces numbered (left), and the spatial arrangement of each interface (right). b, The dimer interfaces formed by equivalent domains in the CLOCK–BMAL1 complex, corresponding to the interfaces 1, 2 and 4 of HIF-2α–ARNT heterodimer. c, The β-sheet-mediated antiparallel interface of isolated HIF-2α–ARNT PAS-B complex with mutations. d, The crystal structure showing the interaction between ARNT and TACC3 (left), and its superimpositions with HIF-2α–ARNT complex through the PAS-B domain of ARNT in two views (right). Colours used in labels match those used in figures for the same components. The binding position of TACC3 peptide with respect to ARNT PAS-B cannot be fully accommodated in our quaternary structure, as some steric clashes would result involving TACC3 and the HIF-2α PAS-B domain.

Extended Data Figure 5 Ligand binding sites in the HIF-2α–ARNT complex.

a, Acriflavine is a mixture of proflavine and trypaflavin. b, Binding tests of acriflavine, proflavine and trypaflavin to protein complexes of HIF-2α–ARNT (left) and HIF-1α–ARNT (right). The calculated Kd values for acriflavine, proflavine and trypaflavin were 41 nM, 31 nM and 34 nM for HIF-2α–ARNT, and 40 nM, 29 nM and 56 nM for HIF-1α–ARNT, respectively. Representative data of at least two experiments, shown as mean ± s.d. from three technical replicates. c, A proposed mechanism by which 0X3 binding can destabilize the HIF-2α–ARNT heterodimer. Shown is the proximity of 0X3 within the PAS-B domain of HIF-2α and R366 from the PAS-B domain of ARNT. 0X3 binding can potentially influence domain–domain interactions mediated by R366. We showed in Fig. 2b that R366 is a highly sensitive site for maintaining the stability of HIF-2α–ARNT heterodimer. 0X3 binding to the PAS-B domain of HIF-2α could further influence interfaces 3, 4 and 5 (shown in Extended Data Fig. 4a). d–g, 0X3 (d) binds at the inner pocket of HIF-2α PAS-B domain, while proflavine (f) binds at the interfacial pocket formed by ARNT PAS-A and HIF-2α PAS-B domains; corresponding positions in the structures of HIF-1α PAS-B (e) and HIF-1α–ARNT–DNA complex (g) are also shown. The yellow dotted lines represent hydrogen bonds. h, Pocket volumes calculated with CASTp program58 at default 1.4 Å probe radius.

Extended Data Figure 6 Interactions between the HIF-2α–ARNT complex and DNA.

a, Recognition of the HRE site by the bHLH domains. Overall structure of HIF-2α–ARNT–DNA with each domain labelled (left); and detailed interactions between the core HRE site (blue) and the bHLH domains of HIF-2α (upper right) or ARNT (lower right) are shown, with grey meshes showing 2Fo Fc electron density contoured at 0.8σ. Hydrogen bonds (2.5–3.5 Å) are indicated by the brown dotted lines, while hydrophobic contact (3.6 Å) is shown by the green one. b, Schematic recognition diagram of HIF-2α–ARNT to the HRE sequence (blue) on DNA. The brown and green dotted lines represent hydrogen bonds and hydrophobic contact, respectively. The black arrow indicates the nucleotide interacting with residues N184 and K186 from HIF-2α PAS-A domain. Additional basic residues from HIF-2α and ARNT bHLH domains (HIF-2α K16, K18, R20, R24, R26 and ARNT R91, R99, R101) that can interact with DNA (mainly through the phosphate backbone) are also labelled in Extended Data Fig. 9. c, HRE DNA binding assay of HIF-2α–ARNT protein complex in wild type (WT) or point-mutated forms using fluorescence polarization. Representative data of at least two experiments are shown as mean ± s.d. from three technical replicates. Calculated approximate Kd values are shown in parentheses. d, The interactions between HIF-2α PAS-A domain and DNA, with the GH loop (including residues N184 and K186) and interacting nucleotides meshed by 2Fo Fc map at 0.8σ.

Extended Data Figure 7 Locations of cancer-related missense mutations on the HIF-α–ARNT heterodimers.

a, Detailed information of the selected cancer-related mutations in HIF-2α and HIF-1α. The information about tissue and histology was adopted from the COSMIC database48 and other publications45,59,60,61,62. b, c, Spatial distribution of the HIF-2α (b) and HIF-1α (c) mutations in the heterodimers. Arrows point to close-up views of several regions in each heterodimer. The sequence positions of these mutations are also labelled in Extended Data Fig. 9.

Extended Data Figure 8 Spatial positions of phosphorylation sites at the HIF-α PAS-B domains in the context of HIF-α–ARNT complexes.

The phosphorylation sites T324 of HIF-2α (a) and S247 of HIF-1α (b) are shown as yellow sticks. Their corresponding residues T322 of HIF-1α and S249 of HIF-2α are also shown. Colours used in labels match those used in figures for the same components. HIF-2α is phosphorylated at residue T324 by protein kinase D1, but the equivalent residue in HIF-1α (T322) cannot be phosphorylated49. The differential positioning of their threonine residues next to a non-conserved loop between PAS-A and PAS-B domains may explain why they cannot be similarly phosphorylated. Casein kinase 1 (CK1) can phosphorylate HIF-1α at S247 (ref. 50). We predict that the equivalent residue S249 in HIF-2α may also be the target of CK1 (ref. 9), since the local environments for these residues are indistinguishable, with both residues being solvent accessible.

Extended Data Figure 9 Comparison of mouse ARNT, BMAL1, CLOCK, HIF-1α and HIF-2α proteins.

Sequence alignment of these five proteins includes the bHLH, PAS-A and PAS-B domains. Alignment was conducted by ClustalW263 and then processed by ESPript 3.064. The secondary structures of each domain of HIF-2α–ARNT complex, and residues involved in domain interfaces, protein–DNA or protein–compound interactions, are differently labelled above (for ARNT) or below (for HIF-2α) the sequences. In addition, the HIF-2α (magenta) or HIF-1α (blue) residues with cancer-related mutations (mainly selected from the COSMIC database48) and phosphorylation sites at the PAS-B domains are also labelled, further below the secondary structures of HIF-2α.

Extended Data Table 1 Data collection and refinement statistics

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Wu, D., Potluri, N., Lu, J. et al. Structural integration in hypoxia-inducible factors. Nature 524, 303–308 (2015). https://doi.org/10.1038/nature14883

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